The development of effective T cell based immunotherapy for cancer, either in the form of a therapeutic vaccine or as adoptive T cell therapy continues to be one of the major areas of research in the field of tumor immunology. Our laboratory has recently designed a novel immunization strategy (TriVax), consisting of synthetic peptides corresponding to CD8 T cell epitopes, Poly-IC adjuvant (a Toll-like receptor agonist) and immune costimulatory antibodies. Using a mouse model of malignant melanoma we observed that TriVax was capable of inducing large antigen-specific, tumor reactive CD8 T cell responses that can amount to more than 50% of the total CD8 T cells. When evaluating the therapeutic effects of TriVax against established B16 melanomas, we observed that the anti-tumor effects (tumor rejection and increased survival) were significantly better in the absence of interferon-gamma (IFN?). These observations are paradoxical since IFN? has long been considered to be an effector cytokine that provides strong anti-tumor effects. Further in vitro experiments have revealed that vaccine generated CD8 T cells were able to recognize B16 melanoma cells less effectively when the tumor cells were treated with IFN? supporting our in vivo anti-tumor observations. These results are puzzling since treatment of B16 melanoma cells with IFN? increases significantly the expression of MHC-I molecules, which should augment, not decrease the tumor's T cell antigenicity. We hypothesize that the specific peptide/MHC-I complexes (cognate MHC-I) recognized by the T cells are not increased to the same extent as the irrelevant peptide/MHC-I (non-cognate) complexes after treatment with IFN?, reducing the overall specific epitope density and leading to a decrease in the antigenicity of tumor cells. In addition, we hypothesize that excess of non-cognate MHC-I on tumor cells prevents the full activation of the T cells by reducing the ability of the CD8 molecules to recruit the Lck lymphocyte-specific tyrosine kinase into the immunological synapse. Furthermore, exposure of tumors to IFN? enhances the expression of ligands for CD8 T cell inhibitory receptors (PD1 and LAG3), which are also detrimental for the therapeutic response to TriVax immunotherapy. The two main objectives of this application will be to clarify the mechanisms by which IFN? exerts a negative effect in the therapeutic efficacy of CD8 T cell immunotherapy and to investigate means to overcome these obstacles in order to develop more effective therapeutic strategies for established malignancies. To test our hypotheses and fulfill these objectives we propose to study the following specific aims: 1) To investigate the mechanisms by which IFN? reduces the antigenicity of melanoma for CD8 T lymphocytes; 2) To assess the importance of the IFN? induced expression of PD1 and LAG3 ligands on tumor cells for the therapeutic efficacy of TriVax against melanoma. 3) To explore several strategies to enhance the therapeutic efficacy of TriVax by overcoming the negative effects of IFN?; and 4) To evaluate the negative effects of IFN? using TriVax in a mouse model of breast cancer. The results of these studies will help clarify the dual role that IFN? appears to play in T cell based tumor immunotherapy and will allow us to design more effective therapeutic strategies for established cancers. PUBLIC HEALTH RELEVANCE: One of the major obstacles for developing effective vaccines for treating cancer has been producing vaccines that induce strong immune responses against tumors. Unfortunately, most current vaccine types generate minimal immune responses and have little effect against established tumors. We have designed a novel vaccination approach called TriVax that utilizes 3 basic components: 1) synthetic peptides (protein fragments) derived from tumor antigens that stimulate T lymphocytes; 2) potent immunological adjuvants that activate the immune system; and 3) immune stimulatory monoclonal antibodies that enhance the efficacy of T cells to react with tumor cells. Preliminary results in a mouse model of malignant melanoma using TriVax demonstrates that this strategy is effective in inducing strong anti-tumor-T cell responses capable of decreasing tumor growth and enhancing survival. Interestingly, we also observed that TriVax is a substantially better therapy for established tumors, resulting in total tumor eradications in mice that are deficient of interferon-gamma. These results are confounding because interferon-gamma is a cytokine considered to be beneficial for immunotherapy against cancer. The goal of proposed studies is to undercover the mechanisms involved by which interferon-gamma may be inhibiting the therapeutic efficacy of TriVax and to develop strategies to overcome the negative effects of this cytokine in the treatment of cancer using optimized vaccines. The results from these studies will serve as preclinical data that will allow us to take this novel approach into the clinic to treat human patients with various types of cancer.